Studies from a diversity of prokaryotic and eukaryotic organisms suggest a gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, plant, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. A comparison of gene sequences with known structure and/or function from one plant species, for example, Arabidopsis thaliana, with those from other plants, allows researchers to develop models for manipulating a plant's traits and developing varieties with valuable properties.
A plant's traits may be controlled through a number of cellular processes. One important way to manipulate that control is through transcription factors proteins that influence the expression of a particular gene or sets of genes. Because transcription factors are key controlling elements of biological pathways, altering the expression levels of one or more transcription factors can change entire biological pathways in an organism. Strategies for manipulating a plant's biochemical, developmental, or phenotypic characteristics by altering a transcription factor expression can result in plants and crops with new and/or improved commercially valuable properties, including traits that improve yield under non-stressed conditions, or survival and yield during periods of abiotic stress. Examples of the latter include, for example, germination in cold conditions, and osmotic stresses such as desiccation, drought, excessive heat, and salt stress.
Desirability of increasing biomass. The ability to increase the biomass or size of a plant would have several important commercial applications. Crop species may be generated that produce larger cultivars, generating higher yield in, for example, plants in which the vegetative portion of the plant is edible.
Increased leaf size may be of particular interest. Increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in total plant photosynthesis is typically achieved by increasing leaf area of the plant. Additional photosynthetic capacity may be used to increase the yield derived from particular plant tissue, including the leaves, roots, fruits or seed, or permit the growth of a plant under decreased light intensity or under high light intensity.
Modification of the biomass of another tissue, such as root tissue, may be useful to improve a plant's ability to grow under harsh environmental conditions, including drought or nutrient deprivation, because larger roots may better reach water or nutrients or take up water or nutrients.
For some ornamental plants, the ability to provide larger varieties would be highly desirable. For many plants, including fruit-bearing trees, trees that are used for lumber production, or trees and shrubs that serve as view or wind screens, increased stature provides improved benefits in the forms of greater yield or improved screening.
Problems associated with drought. A drought is a period of abnormally dry weather that persists long enough to produce a serious hydrologic imbalance (for example crop damage, water supply shortage, etc.). In severe cases, drought can last for many years and have devastating effects on agriculture. Drought is the primary weather-related problem in agriculture and also ranks as one of the major natural disasters of all time, causing not only economic damage, but also loss of human lives. For example, losses from the US drought of 1988 exceeded $40 billion, exceeding those caused by Hurricane Andrew in 1992, the Mississippi River floods of 1993, and the San Francisco earthquake in 1989. The 1984-1985 drought in the Horn of Africa led to a famine that killed 750,000 people.
Problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Drought also causes plants to become more susceptible to various diseases (Simpson (1981) in Water Stress on Plants, (Simpson, G. M., ed.), Praeger, N.Y., pp. 235-265). The most important factor in drought resistance is the ability of the plant to maintain high water status and turgidity, while maintaining carbon fixation. Various adaptive mechanisms influence this ability, including increasing root surface area or depth, osmotic adjustment, and the accumulation of hydrophilic proteins. ABA is also an essential regulatory component of many of these protective features.
Maintaining reproductive performance is another component of yield stability that has been studied in maize. Grain yield is known to be correlated with the kernel number per unit area rather than the weight per kernel. Yield losses in maize due to drought are particularly prevalent when the stress occurs at the transition from vegetative to reproductive growth. A consequence of the growth of maize under drought stress conditions is the delay in silking in relation to pollen shed, adversely affecting kernel set (Edmeades et al. (2000) in Physiology and Modeling Kernel Set in Maize, M. E. Westgate and K. J. Boote, eds (Crop Sci. Soc. America and Amer. Soc. Agron., Madison, Wis.) and reproductive performance. Kernel set is also adversely affected when the grain sink size exceeds the nitrogen uptake capacity from dry soil (Chapman and Edmeades (1999) Crop Sci. 39: 1315-1324). Varieties that were selected for improved yield under drought stress at flowering showed similar performance gains under conditions of low nitrogen, suggesting a common mechanism of tolerance to the two stresses (Beck et al. (1996) in 51st Annual Corn and Sorghum Research Conference, D. Wilkinson, ed (Chicago: ASTA), pp. 85-111; Banzinger et al. (1999) Crop Sci. 39: 1035-1040). When a drought stress occurs between flowering and seed fill of soybeans, total seed yield is reduced due to a reduction in branch growth and thus seed number per branch (Frederick et al. (2001) Crop Sci. 41: 759-763).
Physiological changes occurring in maize plants during drought include:                (a) accumulation of abscisic acid (ABA);        (b) inhibition of cell expansion, resulting in reduced leaf area, reduced silk growth, reduced stem elongation, and reduced root growth;        (c) inhibition of cell division resulting in reduced organ size;        (d) cellular osmotic adjustment (this is more apparent in sorghum and rice and less apparent in maize (Bolanos and Edmeades, 1991)); and        (e) accumulation of proline (during severe drought).        
In addition to the many land regions of the world that are too arid for most, if not all, crop plants, overuse and over-utilization of available water is resulting in an increasing loss of agriculturally-usable land, a process which, in the extreme, results in desertification. The problem is further compounded by increasing salt accumulation in soils, which adds to the loss of available water in soils.
Problems associated with high salt levels. One in five hectares of irrigated land is damaged by salt, an important historical factor in the decline of ancient agrarian societies. This condition is expected to worsen, further reducing the availability of arable land and crop production, since none of the top five food crops—wheat, corn, rice, potatoes, and soybean—can tolerate excessive salt.
Detrimental effects of salt on plants are a consequence of both water deficit resulting in osmotic stress (similar to drought stress) and the effects of excess sodium ions on critical biochemical processes. As with freezing and drought, high saline causes water deficit. The presence of high salt makes it difficult for plant roots to extract water from their environment (Buchanan et al. (2000) in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.). Soil salinity is thus one of the more important variables that determines where a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. To compound the problem, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture. The latter is compounded by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt level in the whole soil profile.
Problems associated with excessive heat. Germination of many crops is very sensitive to temperature. A transcription factor that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates. Seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeleton, and impairs membrane function (Buchanan et al. (2000) supra).
Heat shock may produce a decrease in overall protein synthesis, accompanied by expression of heat shock proteins. Heat shock proteins function as chaperones and are involved in refolding proteins denatured by heat.
Heat stress often accompanies conditions of low water availability. Heat itself is seen as an interacting stress and adds to the detrimental effects caused by water deficit conditions. Evaporative demand exhibits near exponential increases with increases in daytime temperatures, and can result in high transpiration rates and low plant water potentials (Hall et al. (2000) Plant Physiol. 123: 1449-1458). High-temperature damage to pollen almost always occurs in conjunction with drought stress, and rarely occurs under well-watered conditions. It may be difficult to separate the effects of heat and drought stress on pollination and plant metabolism, and thus an understanding of the interaction between these and other stresses may be important when developing strategies to enhance stress tolerance by genetic manipulation.
Problems associated with excessive cold or chilling conditions. The term “chilling sensitivity” has been used to describe many types of physiological damage produced at low, but above freezing, temperatures. Most crops of tropical origins such as soybean, rice, maize and cotton are easily damaged by chilling. Typical cold damage includes wilting, necrosis, chlorosis or leakage of ions from cell membranes. The underlying mechanisms of chilling sensitivity are not completely understood yet, but probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (disruption of photosynthesis due to high light intensities) often occurs under clear atmospheric conditions subsequent to cold late summer/autumn nights. Chilling may lead to yield losses and lower product quality through the delayed ripening of maize. Another consequence of poor growth is the rather poor ground cover of maize fields in spring, often resulting in soil erosion, increased occurrence of weeds, and reduced uptake of nutrients. A retarded uptake of mineral nitrogen could also lead to increased losses of nitrate into the ground water. By some estimates, chilling accounts for monetary losses in the United States (US) behind only to drought and flooding.
Desirability of altered sugar sensing. Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Sucrose, for example, is the major transport form of photosynthate and its flux through cells has been shown to affect gene expression and alter storage compound accumulation in seeds (source-sink relationships). Glucose-specific hexose-sensing has also been described in plants and is implicated in cell division and repression of “famine” genes (photosynthetic or glyoxylate cycles).
Water deficit is a common component of many plant stresses. Water deficit occurs in plant cells when the whole plant transpiration rate exceeds the water uptake. In addition to drought, other stresses, such as salinity and low temperature, produce cellular dehydration (McCue and Hanson (1990) Trends Biotechnol. 8: 358-362).
Salt and drought stress signal transduction consist of ionic and osmotic homeostasis signaling pathways. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. The pathway regulating ion homeostasis in response to salt stress has been reviewed recently by Xiong and Zhu (Xiong and Zhu (2002) Plant Cell Environ. 25: 131-139).
The osmotic component of salt stress involves complex plant reactions that overlap with drought and/or cold stress responses.
Common aspects of drought, cold and salt stress response have been reviewed recently by Xiong and Zhu (2002) supra. Those include:                (a) transient changes in the cytoplasmic calcium levels very early in the signaling event (Knight, (2000) Int. Rev. Cytol. 195: 269-324; Sanders et al. (1999) Plant Cell 11: 691-706);        (b) signal transduction via mitogen-activated and/or calcium dependent protein kinases (CDPKs; see Xiong and Zhu (2002) supra) and protein phosphatases (Merlot et al. (2001) Plant J. 25: 295-303; Tähtiharju and Palva (2001) Plant J. 26: 461-470);        (c) increases in ABA levels in response to stress triggering a subset of responses (Xiong and Zhu (2002) supra, and references therein);        (d) inositol phosphates as signal molecules (at least for a subset of the stress responsive transcriptional changes (Xiong et al. (2001) Genes Dev. 15: 1971-1984));        (e) activation of phospholipases which in turn generate a diverse array of second messenger molecules, some of which might regulate the activity of stress responsive kinases (phospholipase D functions in an ABA independent pathway, Frank et al. (2000) Plant Cell 12: 111-124);        (f) induction of late embryogenesis abundant (LEA) type genes including the CRT/DRE-containing COR/RD genes (Xiong and Zhu (2002) supra);        (g) increased levels of antioxidants and compatible osmolytes such as proline and soluble sugars (Hasegawa et al. (2000) Annu. Rev. Plant Mol. Plant Physiol. 51: 463-499);        (h) accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals (Hasegawa et al. (2000) supra).        
ABA biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and ABA-independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.
Based on the commonality of many aspects of cold, drought and salt stress responses, it can be concluded that genes that increase tolerance to cold or salt stress can also improve drought stress protection. In fact, this has already been demonstrated for transcription factors (in the case of AtCBF/DREB1) and for other genes such as OsCDPK7 (Saijo et al. (2000) Plant J. 23: 319-327), or AVP1 (a vacuolar pyrophosphatase-proton-pump; Gaxiola et al. (2001) Proc. Natl. Acad. Sci. USA 98: 11444-11449).
The present invention relates to methods and compositions for producing transgenic plants with modified traits, particularly traits that address agricultural and food needs. These traits, including increased biomass, altered sugar sensing, and tolerance to abiotic stress, may provide significant value in that greater yield may be achieved, and/or the plant can then thrive in hostile environments, where, for example, high or low temperature, low water availability or high salinity may limit or prevent growth of non-transgenic plants.
We have identified polynucleotides encoding transcription factors, including G1073 (atHRC1), and equivalogs in the G1073 clade of transcription factor polypeptides, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for their biomass and tolerance to abiotic stresses. In so doing, we have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.